Aptamers are oligonucleic acid or peptide molecules that are characterized by specific binding to a target molecule. The specific binding underlies the ability of aptamers to act as potent modifiers of protein function. Although they are now known to occur naturally in riboswitches, aptamers as originally described are engineered molecules produced through iterative selection processes drawing from a large random sequence pool. Aptamers are useful in basic research and hold special promise for clinical applications as macromolecular drugs.
A nucelic acid aptamer is essentially a single-stranded oligonucleotide (DNA or RNA), or a series thereof. Selection processes for producing nucleic acid aptamers include a combinatorial technique known as SELEX (“Systematic Evolution of Ligands by Exponential Enrichment”; Tuerk C. & Gold L., SCIENCE (1990), 249 (4968): 505-510), and the process known as in vitro selection in which RNA ligands are selected against various organic dyes (Ellington A. D. & Szostak J. W., NATURE (1990), 346: 818-822). In any case, the resulting aptamers bind tightly and selectively to their ligands and may potentially be employed in targeted molecular therapies.
Certain aptamers and their molecular ligands have been tested for potential therapeutic applications. In particular, the FDA has approved clinical trials for Macugen™ (pegaptinib sodium), an aptamer with application in ophthalmic pathologies. DNA and RNA aptamers also have been generated against HIV-1 proteins to target viral enzymes (Reverse Transcriptase, Protease, Integrase) or viral expression (Rev, Tat), packaging and entry (Gag, nucleocapsid, gp120). For example, certain RNA aptamers against HIV-1 Reverse Transcriptase have been isolated and tested in vitro and in vivo, and some single-stranded DNA aptamers against HIV-1 Reverse Transcriptase have also been described.
A “G-quadruplex” (also known as a G-tetrad or G4-DNA) is a four-stranded nucleic acid structure formed from a sequence that is guanine-rich and thus capable of forming a square arrangement of guanines (a tetrad), which is stabilized by Hoogsteen hydrogen bonding and further stabilized by the existence of a monovalent cation (especially potassium) in the center of the tetrads. A G-quadruplex can be formed of DNA, RNA, LNA and PNA, and may be intramolecular, bimolecular or tetramolecular. Depending on the direction of the strands or parts of a strand that form the tetrads, structures may be described as parallel or antiparallel. Potential quadruplex sequences have been identified in eukaryotic telomeres. Recently, non-telomeric quadruplexes have been identified, e.g. in the proto-oncogene c-myc, H-ras, N-ras promoter regions. Thus, quadruplex structures may be a common control element of gene expression. Increasing interest exists in finding and identifying small molecules and naturally occurring proteins that may be control targets of G-quadruplex structures and thus may be candidates for specific therapeutic interventions. The SELEX technique for generating aptamers has been used to generate a few sequence variants that produce variants of the G-quadruplex structure.
G-rich oligonucleotides (GRO), a novel class of antiproliferative agents, have also been described. The DNA aptamer AGRO100 is an experimental anticancer drug that has entered human clinical trials. It is a non-antisense, guanosine-rich phosphodiester oligodeoxynucleotide that also forms stable G-quadruplex structures. The biological activity of GROs results from their specific binding to specific cellular proteins as aptamers. Nucleolin has is an important target protein of GROs, and is a multifunctional protein expressed at high levels by cancer cells.
Certain DNA oligomers having G-quadruplex structures have been described as inhibitors of certain retroviral functions. For example, the DNA oligomers Zintevir™, 93del, and 112 del are different G-quadruplex aptamers possessing anti-HIV activity. Zintevir™ is a unimolecular 17-mer ODN-derived aptamer (AR177, T30177, and T30695) which prevents the binding of HIV gp120 to CD4 cells and inhibits HIV integrase, and is among the first oligonucleotides to enter human clinical trials. 93 del and 112 del are dimeric DNA G-quadruplex aptamers (shorter DNA aptamers derived from ODN93 and ODN112) originally selected as inhibitors against RNaseH activity).
Thus, the quadruplex structure may be important structural component of new antiviral and ant-cancer drugs, and may be useful in the development of strategies for designing new anti-viral and anti-cancer drugs, particularly for combating the immunodeficiency viruses including HIV-1, HIV-2 and SIV. Consequently, there is a need in the art for methods that allow the identification of aptamers that inhibit proteins that are critical to HIV-1, HIV-2 and SIV replication, as well as specific aptamers that recognize these molecules.